Publications by authors named "Stephen M Hewitt"

352 Publications

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

JCI Insight 2022 Jul 19. Epub 2022 Jul 19.

Facultad de Medicina Clínica Alemana de Santiago, Universidad del Desarrollo, Santiago, Chile.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.
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http://dx.doi.org/10.1172/jci.insight.160332DOI Listing
July 2022

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Am J Respir Crit Care Med 2022 Jul 11. Epub 2022 Jul 11.

University of North Carolina at Chapel Hill School of Medicine, 6797, Marsico Lung Institute, Chapel Hill, North Carolina, United States;

Rationale: The incidence and sites of mucus accumulation, and molecular regulation of mucin gene expression, in COVID-19 lung disease have not been reported.

Objectives: Characterize incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease.

Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by AB-PAS and immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. SARS-CoV-2-infected human bronchial epithelial (HBE) cultures were utilized to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures.

Measurements And Main Results: MUC5B and variably MUC5AC RNA levels were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the sub-acute/chronic disease phase following SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of COVID-19 subjects in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days post inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days post inoculation. SARS-CoV-2 infection of HBE cultures induced expression of EGFR ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways, or dexamethasone administration, reduced SARS-CoV-2-induced mucin expression.

Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation post SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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http://dx.doi.org/10.1164/rccm.202111-2606OCDOI Listing
July 2022

Advancing Research on Medical Image Perception by Strengthening Multidisciplinary Collaboration.

JNCI Cancer Spectr 2022 01;6(1)

Department of Radiology, Harvard Medical School, Boston, MA, USA.

Medical image interpretation is central to detecting, diagnosing, and staging cancer and many other disorders. At a time when medical imaging is being transformed by digital technologies and artificial intelligence, understanding the basic perceptual and cognitive processes underlying medical image interpretation is vital for increasing diagnosticians' accuracy and performance, improving patient outcomes, and reducing diagnostician burnout. Medical image perception remains substantially understudied. In September 2019, the National Cancer Institute convened a multidisciplinary panel of radiologists and pathologists together with researchers working in medical image perception and adjacent fields of cognition and perception for the "Cognition and Medical Image Perception Think Tank." The Think Tank's key objectives were to identify critical unsolved problems related to visual perception in pathology and radiology from the perspective of diagnosticians, discuss how these clinically relevant questions could be addressed through cognitive and perception research, identify barriers and solutions for transdisciplinary collaborations, define ways to elevate the profile of cognition and perception research within the medical image community, determine the greatest needs to advance medical image perception, and outline future goals and strategies to evaluate progress. The Think Tank emphasized diagnosticians' perspectives as the crucial starting point for medical image perception research, with diagnosticians describing their interpretation process and identifying perceptual and cognitive problems that arise. This article reports the deliberations of the Think Tank participants to address these objectives and highlight opportunities to expand research on medical image perception.
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http://dx.doi.org/10.1093/jncics/pkab099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8826981PMC
January 2022

Protein expression of the gp78 E3 ligase predicts poor breast cancer outcome based on race.

JCI Insight 2022 07 8;7(13). Epub 2022 Jul 8.

Department of Pathology and Cell Biology, Columbia University Irvine Medical Center, New York, New York, USA.

Women of African ancestry suffer higher rates of breast cancer mortality compared with all other groups in the United States. Though the precise reasons for these disparities remain unclear, many recent studies have implicated a role for differences in tumor biology. Using an epitope-validated antibody against the endoplasmic reticulum-associated E3 ligase, gp78, we show that elevated levels of gp78 in patient breast cancer cells predict poor survival. Moreover, high levels of gp78 are associated with poor outcomes in both ER+ and ER- tumors, and breast cancers expressing elevated amounts of gp78 protein are enriched in gene expression pathways that influence cell cycle, metabolism, receptor-mediated signaling, and cell stress response pathways. In multivariate analysis adjusted for subtype and grade, gp78 protein is an independent predictor of poor outcomes in women of African ancestry. Furthermore, gene expression signatures, derived from patients stratified by gp78 protein expression, are strong predictors of recurrence and pathological complete response in retrospective clinical trial data and share many common features with gene sets previously identified to be overrepresented in breast cancers based on race. These findings implicate a prominent role for gp78 in tumor progression and offer insights into our understanding of racial differences in breast cancer outcomes.
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http://dx.doi.org/10.1172/jci.insight.157465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9310521PMC
July 2022

Locoregional tumor burden and risk of mortality in metastatic breast cancer.

NPJ Precis Oncol 2022 Apr 5;6(1):22. Epub 2022 Apr 5.

ELIASSEN Group, Reston, VA, USA.

The role of lymph node involvement and tumor size in metastatic disease including breast cancer is unclear. Here, nodal metastasis and T stage on the risk of mortality were investigated in de novo metastatic breast cancer population (35812 patients) in the Surveillance, Epidemiology, and End Results (SEER) Program database in the United States. We found an association between all-cause mortality and regional node involvement (adjusted hazard ratio [HR] = 1.45, 95% confidence interval [CI] 1.36-1.55, p < 0.0001) or T stage (HR = 1.20, 95% CI 1.14-1.25, p < 0.0001), independent of known clinicopathologic measurements. Number of positive nodes, and size and chest wall involvement of the breast tumors exhibited similar significance for breast cancer-specific mortality in the population (p < 0.0001 each), and all-cause mortality in hormone receptor (HR)-positive/HER2-negative (HR/HER2), HR/HER2, HR/HER2 and triple-negative metastatic breast cancer subtypes. Thus, nodal involvement and T stage are independent risk factors for mortality in the population of de novo metastatic breast cancer.
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http://dx.doi.org/10.1038/s41698-022-00265-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8983737PMC
April 2022

Reproducible, high-dimensional imaging in archival human tissue by multiplexed ion beam imaging by time-of-flight (MIBI-TOF).

Lab Invest 2022 07 29;102(7):762-770. Epub 2022 Mar 29.

Department of Pathology, Stanford University, Stanford, CA, 94304, USA.

Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute's (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R = 0.94 ± 0.04) and frequency (R = 0.95 ± 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R = 0.85 ± 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between serial sections yielded an average correlation of R = 0.94 ± 0.05. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.
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http://dx.doi.org/10.1038/s41374-022-00778-8DOI Listing
July 2022

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19.

medRxiv 2022 Mar 3. Epub 2022 Mar 3.

Programa de Inmunogenética e Inmunología Traslacional, Instituto de Ciencias e Innovación en Medicina, Facultad de Medicina Clínica Alemana, Universidad del Desarrollo, Santiago, Chile.

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including MIS-C and chilblain-like lesions (CLL), otherwise known as "COVID toes", remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Summary: NET formation and degradation are dysregulated in pediatric and symptomatic adult patients with various complications of COVID-19, in association with disease severity. NET degradation impairments are multifactorial and associated with natural inhibitors of DNase 1, G-actin and anti-DNase1L3 and anti-NET antibodies. Infection with the Omicron variant is associated with decreased levels of NETs when compared to other SARS-CoV-2 strains.
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http://dx.doi.org/10.1101/2022.02.24.22271475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8902885PMC
March 2022

The Impact of the COVID-19 Pandemic on the Scientific Community.

Authors:
Stephen M Hewitt

J Histochem Cytochem 2022 04 28;70(4):271-272. Epub 2022 Feb 28.

Journal of Histochemistry & Cytochemistry, Truchas, New Mexico.

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http://dx.doi.org/10.1369/00221554221084760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8971686PMC
April 2022

Single-cell biology uncovers apoptotic cell death and its spatial organization as a potential modifier of tumor diversity in HCC.

Hepatology 2022 Sep 10;76(3):599-611. Epub 2022 Feb 10.

Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

Background And Aims: HCC is a highly aggressive and heterogeneous cancer type with limited treatment options. Identifying drivers of tumor heterogeneity may lead to better therapeutic options and favorable patient outcomes. We investigated whether apoptotic cell death and its spatial architecture is linked to tumor molecular heterogeneity using single-cell in situ hybridization analysis.

Approach And Results: We analyzed 254 tumor samples from two HCC cohorts using tissue microarrays. We developed a mathematical model to quantify cellular diversity among HCC samples using two tumor markers, cyclin-dependent kinase inhibitor 3 and protein regulator of cytokinesis 1 as surrogates for heterogeneity and caspase 3 (CASP3) as an apoptotic cell death marker. We further explored the impact of potential dying-cell hubs on tumor cell diversity and patient outcome by density contour mapping and spatial proximity analysis. We also developed a selectively controlled in vitro model of cell death using CRISPR/CRISPR-associated 9 to determine therapy response and growth under hypoxic conditions. We found that increasing levels of CASP3 tumor cells are associated with higher tumor diversity. Interestingly, we discovered regions of densely populated CASP3 , which we refer to as CASP3 cell islands, in which the nearby cellular heterogeneity was found to be the greatest compared to cells farther away from these islands and that this phenomenon was associated with survival. Additionally, cell culture experiments revealed that higher levels of cell death, accompanied by increased CASP3 expression, led to greater therapy resistance and growth under hypoxia.

Conclusions: These results are consistent with the hypothesis that increased apoptotic cell death may lead to greater tumor heterogeneity and thus worse patient outcomes.
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http://dx.doi.org/10.1002/hep.32345DOI Listing
September 2022

Evidence of SARS-CoV-2-Specific T-Cell-Mediated Myocarditis in a MIS-A Case.

Front Immunol 2021 9;12:779026. Epub 2021 Dec 9.

Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, United States.

A 26-year-old otherwise healthy man died of fulminant myocarditis. Nasopharyngeal specimens collected premortem tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Histopathological evaluation of the heart showed myocardial necrosis surrounded by cytotoxic T-cells and tissue-repair macrophages. Myocardial T-cell receptor (TCR) sequencing revealed hyper-dominant clones with highly similar sequences to TCRs that are specific for SARS-CoV-2 epitopes. SARS-CoV-2 RNA was detected in the gut, supporting a diagnosis of multisystem inflammatory syndrome in adults (MIS-A). Molecular targets of MIS-associated inflammation are not known. Our data indicate that SARS-CoV-2 antigens selected high-frequency T-cell clones that mediated fatal myocarditis.
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http://dx.doi.org/10.3389/fimmu.2021.779026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8695925PMC
January 2022

Mitochondrial C5aR1 activity in macrophages controls IL-1β production underlying sterile inflammation.

Sci Immunol 2021 Dec 24;6(66):eabf2489. Epub 2021 Dec 24.

Laboratory of Pathology, National Cancer Institute (NCI), NIH, Bethesda, MD 20892, USA.

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the “complosome,” functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro–IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.
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http://dx.doi.org/10.1126/sciimmunol.abf2489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8902698PMC
December 2021

Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Front Immunol 2021 11;12:765898. Epub 2021 Nov 11.

Veteran's Administration (VA) Cooperative Studies Program, Veteran's Administration (VA) Boston Healthcare System (151MAV), Jamaica Plain, MA, United States.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.
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http://dx.doi.org/10.3389/fimmu.2021.765898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8632241PMC
February 2022

The tale of TILs in breast cancer: A report from The International Immuno-Oncology Biomarker Working Group.

NPJ Breast Cancer 2021 Dec 1;7(1):150. Epub 2021 Dec 1.

Department of Pathology, Hospital de Oncología Maria Curie, Buenos Aires, Argentina.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.
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http://dx.doi.org/10.1038/s41523-021-00346-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8636568PMC
December 2021

Defining a (Cancer Stem Cell) Niche.

Authors:
Stephen M Hewitt

J Histochem Cytochem 2021 12;69(12):747

Journal of Histochemistry & Cytochemistry, Truchas, New Mexico, (SMH).

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http://dx.doi.org/10.1369/00221554211061358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8647631PMC
December 2021

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Nat Methods 2022 03 22;19(3):284-295. Epub 2021 Nov 22.

Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA, USA.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.
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http://dx.doi.org/10.1038/s41592-021-01316-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9264278PMC
March 2022

Regulates Chemoresistance in Association With by Inhibiting Apoptosis Pathway in Patients With Cervical Cancer.

Cancer Genomics Proteomics 2021 Nov-Dec;18(6):699-713

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, U.S.A.

Background/aim: Cryptochrome 1 (CRY1), a core circadian gene, modulates circadian rhythm and carcinogenesis. Here, we investigated the role of CRY1 and its correlation with NANOG, a stem cell transcription factor, in cervical cancer.

Materials And Methods: Immunohistochemistry with tissue microarray was performed to evaluate CRY1 and NANOG expression in cervical cancer tissues, and their functional roles were assessed in cervical cancer cell lines.

Results: CRY1 or NANOG was significantly over-expressed in cervical cancer tissues. Notably, combined over-expression of CRY1 and NANOG was correlated with a significantly poor OS and DFS and showed a stronger predictive value for chemoradiation response than each single protein. Furthermore, siCRY1 induced apoptosis, decreased NANOG expression, suppressed STAT3 signalling, and activated p53 signalling in cervical cancer cell lines.

Conclusion: CRY1 and NANOG over-expression serves as a strong predictive biomarker for prognosis and chemoradiation response, and may be a new therapeutic target in patients with cervical cancer.
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http://dx.doi.org/10.21873/cgp.20291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569817PMC
February 2022

Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition.

Cancers (Basel) 2021 Jul 30;13(15). Epub 2021 Jul 30.

Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA 98109, USA.

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.
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http://dx.doi.org/10.3390/cancers13153843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345163PMC
July 2021

Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions.

JCI Insight 2021 09 8;6(17). Epub 2021 Sep 8.

Division of Cancer Prevention and.

BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.
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http://dx.doi.org/10.1172/jci.insight.147096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492350PMC
September 2021

Multiplex Tissue Imaging Harmonization: A Multicenter Experience from CIMAC-CIDC Immuno-Oncology Biomarkers Network.

Clin Cancer Res 2021 09 12;27(18):5072-5083. Epub 2021 Jul 12.

Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Purpose: The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) network supported by the NCI Cancer Moonshot initiative was established to provide correlative analyses for clinical trials in cancer immunotherapy, using state-of-the-art technology. Fundamental to this initiative is implementation of multiplex IHC assays to define the composition and distribution of immune infiltrates within tumors in the context of their potential role as biomarkers. A critical unanswered question involves the relative fidelity of such assays to reliably quantify tumor-associated immune cells across different platforms.

Experimental Design: Three CIMAC sites compared across their laboratories: (i) image analysis algorithms, (ii) image acquisition platforms, (iii) multiplex staining protocols. Two distinct high-dimensional approaches were employed: multiplexed IHC consecutive staining on single slide (MICSSS) and multiplexed immunofluorescence (mIF). To eliminate variables potentially impacting assay performance, we completed a multistep harmonization process, first comparing assay performance using independent protocols followed by the integration of laboratory-specific protocols and finally, validating this harmonized approach in an independent set of tissues.

Results: Data generated at the final validation step showed an intersite Spearman correlation coefficient () of ≥0.85 for each marker within and across tissue types, with an overall low average coefficient of variation ≤0.1.

Conclusions: Our results support interchangeability of protocols and platforms to deliver robust, and comparable data using similar tissue specimens and confirm that CIMAC-CIDC analyses may therefore be used with confidence for statistical associations with clinical outcomes largely independent of site, antibody selection, protocol, and platform across different sites.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-2051DOI Listing
September 2021

CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice.

Cell Rep Med 2021 06 1;2(6):100297. Epub 2021 Jun 1.

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.
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http://dx.doi.org/10.1016/j.xcrm.2021.100297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233664PMC
June 2021

Clinical Significance of Tumor Infiltrating Lymphocytes in Association with Hormone Receptor Expression Patterns in Epithelial Ovarian Cancer.

Int J Mol Sci 2021 May 27;22(11). Epub 2021 May 27.

Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul 03722, Korea.

Hormone receptor expression patterns often correlate with infiltration of specific lymphocytes in tumors. Specifically, the presence of specific tumor-infiltrating lymphocytes (TILs) with particular hormone receptor expression is reportedly associated with breast cancer, however, this has not been revealed in epithelial ovarian cancer (EOC). Therefore, we investigated the association between hormone receptor expression and TILs in EOC. Here we found that ERα, AR, and GR expression increased in EOC, while PR was significantly reduced and ERβ expression showed a reduced trend compared to normal epithelium. Cluster analysis indicated poor disease-free survival (DFS) in AR+/GR+/PR+ subgroup (triple dominant group); while the Cox proportional-hazards model highlighted the triple dominant group as an independent prognostic factor for DFS. In addition, significant upregulation of FoxP3+ TILs, PD-1, and PD-L1 was observed in the triple dominant group compared to other groups. NanoString analyses further suggested that tumor necrosis factor (TNF) and/or NF-κB signaling pathways were activated with significant upregulation of , , , , , , , and in the triple dominant EOC group. The triple dominant subgroup correlates with poor prognosis in EOC. Moreover, the TNF and/or NF-κB signaling pathways may be responsible for hormone-mediated inhibition of the immune microenvironment.
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http://dx.doi.org/10.3390/ijms22115714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198528PMC
May 2021

Rapidly Progressing Urothelial Carcinoma Due to a Rare TP53 (p.Arg110Pro) Mutation: A Case Report and Review of the Literature.

Res Rep Urol 2021 20;13:181-184. Epub 2021 Apr 20.

Section of Urology, Department of Surgery, The University of Chicago Medicine, Chicago, IL, USA.

We present a case of a 69-year-old male patient diagnosed with high grade (T HG) urothelial carcinoma of the bladder who progressed rapidly towards muscle invasive disease and eventually death despite neoadjuvant chemotherapy and radical cystectomy. We postulate that this may be due to a deleterious underlying somatic gene mutation. Molecular pathologic data obtained on the initial, non-muscle invasive tumor and the final cystectomy specimen, revealed the same TP53 mutation (p.Arg110Pro) in both specimens with a variant allele frequency of 44%. The tumor was tested for 50 common gene mutations in urothelial carcinoma and no other identifiable DNA repair mutations were found, suggesting that this specific TP53 aberration, one that has never been reported in the bladder cancer literature, could be particularly deleterious. Knowing that bladder cancer cell lines that lack TP53 are more resistant to cisplatin and because the tumor lacked any other DNA mutation, this patient may have been a candidate for upfront surgery without neoadjuvant chemotherapy. In addition to histological analysis of the tumor, early molecular and cytogenetic characterization of resected tissue is essential in predicting progression and eventual prognosis of the disease based on identifiable gene mutations. Further comparative prospective studies are required to clarify the importance of molecular heterogeneity and subtyping in bladder cancer.
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http://dx.doi.org/10.2147/RRU.S288948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068478PMC
April 2021

Improving data quality in observational research studies: Report of the Cure Glomerulonephropathy (CureGN) network.

Contemp Clin Trials Commun 2021 Jun 17;22:100749. Epub 2021 Feb 17.

NYU Langone Health, Department of Pediatrics, Division of Nephrology, New York, NY, USA.

Background: High data quality is of crucial importance to the integrity of research projects. In the conduct of multi-center observational cohort studies with increasing types and quantities of data, maintaining data quality is challenging, with few published guidelines.

Methods: The Cure Glomerulonephropathy (CureGN) Network has established numerous quality control procedures to manage the 70 participating sites in the United States, Canada, and Europe. This effort is supported and guided by the activities of several committees, including Data Quality, Recruitment and Retention, and Central Review, that work in tandem with the Data Coordinating Center to monitor the study. We have implemented coordinator training and feedback channels, data queries of questionable or missing data, and developed performance metrics for recruitment, retention, visit completion, data entry, recording of patient-reported outcomes, collection, shipping and accessing of biological samples and pathology materials, and processing, cataloging and accessing genetic data and materials.

Results: We describe the development of data queries and site Report Cards, and their use in monitoring and encouraging excellence in site performance. We demonstrate improvements in data quality and completeness over 4 years after implementing these activities. We describe quality initiatives addressing specific challenges in collecting and cataloging whole slide images and other kidney pathology data, and novel methods of data quality assessment.

Conclusions: This paper reports the CureGN experience in optimizing data quality and underscores the importance of general and study-specific data quality initiatives to maintain excellence in the research measures of a multi-center observational study.
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http://dx.doi.org/10.1016/j.conctc.2021.100749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039553PMC
June 2021

Crosstalk between WNT and STAT3 is mediated by galectin-3 in tumor progression.

Gastric Cancer 2021 09 8;24(5):1050-1062. Epub 2021 Apr 8.

Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.

Background: Aberrant activation of the WNT/β-catenin and STAT3 signaling pathways plays a critical role in cancer progression. However, direct targeting of these pathways as an anti-cancer therapeutic approach needs to be reconsidered due to its serious side effects. Here, we demonstrate that overexpression of WNT induces STAT3 activation in a galectin-3-dependent manner.

Methods: We investigated how galectin-3 mediates the crosstalk between WNT/β-catenin and STAT3 signaling and whether inhibition of galectin-3 can reduce gastric cancer. The molecular mechanisms were analyzed by biochemical assays using cultured gastric cancer cells, patient tissues, and genetically engineered mice. Moreover, we confirm of therapeutic effects of GB1107, a cell-penetrating galectin-3 specific inhibitor, using orthotopic gastric cancer-bearing mice RESULTS: Increased levels of galectin-3 and STAT3 phosphorylation were detected in the stomach tissues of WNT1-overexpressing mouse models. Also, high expression levels and co-localization of β-catenin, pSTAT3, and galectin-3 in patients with advanced gastric cancer were correlated with a poorer prognosis. Galectin-3 depletion significantly decreased STAT3 Tyr705 phosphorylation, which regulates its nuclear localization and transcriptional activation. A peptide of galectin-3 (Y45-Q48) directly bound to the STAT3 SH2 domain and enhanced its phosphorylation. GB1107, a specific membrane-penetrating inhibitor of galectin-3, significantly reduced the activation of both STAT3 and β-catenin and inhibited tumor growth in orthotopic gastric cancer-bearing mice.

Conclusions: We propose that galectin-3 mediates the crosstalk between the WNT and STAT3 signaling pathways. Therefore GB1107, a galectin-3-specific inhibitor, maybe a potent agent with anti-gastric cancer activity. Further studies are needed for its clinical application in gastric cancer therapy.
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http://dx.doi.org/10.1007/s10120-021-01186-5DOI Listing
September 2021

SARS-CoV-2 infection of the oral cavity and saliva.

Nat Med 2021 05 25;27(5):892-903. Epub 2021 Mar 25.

Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.
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http://dx.doi.org/10.1038/s41591-021-01296-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240394PMC
May 2021

INFORM: INFrared-based ORganizational Measurements of tumor and its microenvironment to predict patient survival.

Sci Adv 2021 02 3;7(6). Epub 2021 Feb 3.

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

The structure and organization of a tumor and its microenvironment are often associated with cancer outcomes due to spatially varying molecular composition and signaling. A persistent challenge is to use this physical and chemical spatial organization to understand cancer progression. Here, we present a high-definition infrared imaging-based organizational measurement framework (INFORM) that leverages intrinsic chemical contrast of tissue to label unique components of the tumor and its microenvironment. Using objective and automated computational methods, further, we determine organization characteristics important for prediction. We show that the tumor spatial organization assessed with this framework is predictive of overall survival in colon cancer that adds to capability from clinical variables such as stage and grade, approximately doubling the risk of death in high-risk individuals. Our results open an all-digital avenue for measuring and studying the association between tumor spatial organization and disease progression.
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http://dx.doi.org/10.1126/sciadv.abb8292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857685PMC
February 2021

Kaiso (ZBTB33) subcellular partitioning functionally links LC3A/B, the tumor microenvironment, and breast cancer survival.

Commun Biol 2021 02 1;4(1):150. Epub 2021 Feb 1.

Department of Pathology and Cell Biology, Columbia University Irvine Medical Center, New York, NY, USA.

The use of digital pathology for the histomorphologic profiling of pathological specimens is expanding the precision and specificity of quantitative tissue analysis at an unprecedented scale; thus, enabling the discovery of new and functionally relevant histological features of both predictive and prognostic significance. In this study, we apply quantitative automated image processing and computational methods to profile the subcellular distribution of the multi-functional transcriptional regulator, Kaiso (ZBTB33), in the tumors of a large racially diverse breast cancer cohort from a designated health disparities region in the United States. Multiplex multivariate analysis of the association of Kaiso's subcellular distribution with other breast cancer biomarkers reveals novel functional and predictive linkages between Kaiso and the autophagy-related proteins, LC3A/B, that are associated with features of the tumor immune microenvironment, survival, and race. These findings identify effective modalities of Kaiso biomarker assessment and uncover unanticipated insights into Kaiso's role in breast cancer progression.
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http://dx.doi.org/10.1038/s42003-021-01651-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851134PMC
February 2021

MEK/ERK signaling is a critical regulator of high-risk human papillomavirus oncogene expression revealing therapeutic targets for HPV-induced tumors.

PLoS Pathog 2021 01 22;17(1):e1009216. Epub 2021 Jan 22.

Department of Molecular Genetics & Microbiology, The University of New Mexico School of Medicine, Albuquerque, NM, United States of America.

Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or "high-risk" HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.
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http://dx.doi.org/10.1371/journal.ppat.1009216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857559PMC
January 2021

Network for Biomarker Immunoprofiling for Cancer Immunotherapy: Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC).

Clin Cancer Res 2021 09 8;27(18):5038-5048. Epub 2021 Jan 8.

The Emmes Company, LLC, Rockville, Maryland.

Purpose: Immunoprofiling to identify biomarkers and integration with clinical trial outcomes are critical to improving immunotherapy approaches for patients with cancer. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assay performance further limits comparison and interpretation of data across studies and laboratories.

Experimental Design: To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer Moonshot Initiative of the National Cancer Institute (NCI) and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the NIH.

Results: The CIMAC-CIDC Network is composed of four academic centers with multidisciplinary expertise in cancer immunotherapy that perform validated and harmonized assays for immunoprofiling and conduct correlative analyses. A data coordinating center (CIDC) provides the computational expertise and informatics platforms for the storage, integration, and analysis of biomarker and clinical data.

Conclusions: This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure, validation and harmonization of core immunoprofiling assays, platforms for data ingestion and integration, and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole-exome sequencing, RNA sequencing, cytometry by time of flight, and IHC/immunofluorescence.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8491462PMC
September 2021

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis.

Int J Radiat Oncol Biol Phys 2021 06 30;110(2):526-538. Epub 2020 Dec 30.

Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Electronic address:

Purpose: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging.

Methods And Materials: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63).

Results: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 μg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis.

Conclusions: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.
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http://dx.doi.org/10.1016/j.ijrobp.2020.12.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784947PMC
June 2021
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